CN109820032A - Purposes of the UBE2 inhibitor as food preservation by low temperature agent - Google Patents
Purposes of the UBE2 inhibitor as food preservation by low temperature agent Download PDFInfo
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- CN109820032A CN109820032A CN201910241065.8A CN201910241065A CN109820032A CN 109820032 A CN109820032 A CN 109820032A CN 201910241065 A CN201910241065 A CN 201910241065A CN 109820032 A CN109820032 A CN 109820032A
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Abstract
Purposes the present invention provides the inhibitor of ubiquitin conjugation E2 enzyme (Ubiquitin-conjugating E2 enzyme, UBE2) as food preservation by low temperature agent.The present invention also provides a kind of low-temp antistaling methods of straw mushroom, straw mushroom are placed in the solution of UBE2 inhibitor and are impregnated, then drained away the water, refrigerated, and can obviously delay low temperature to the frostbite of straw mushroom.The present invention matches UBE2 inhibitor at solution, carries out straw mushroom fructification immersion test, finds the frostbite that the inhibitor can obviously delay low temperature to straw mushroom, can be used as the preservation by low temperature agent of the food such as straw mushroom.
Description
Technical field
The invention belongs to field of food, are related to a kind of food preservation technology, specifically UBE2 inhibitor is low as food
The purposes of warm antistaling agent.
Background technique
Straw mushroom (Volvariella volvacea) it is referred to as " Chinese mushroom ", it is a kind of important edible mushroom.Straw mushroom is not only
Delicious flavour, and there is important medical care effect, such as anti-oxidant, adjusting is immunized, is antitumor.Straw mushroom factory culture
It is one of principal item of degrading straw mostly using straw, waste cotton as main matrix.China's agricultural crop straw produces about 800,000,000 per year
Ton.Great economic benefit possessed by straw mushroom degrading straw is by power-assisted resource-conserving, environmentally friendly modernization new agricultural
Development.Straw mushroom delicious meat and growth cycle is short (about 10 days or so), should become the very high edible mushroom of economic benefit.So
And the growth of straw mushroom mycelia requires relatively high temperature (28-35 °C) with solid, especially its mycelia and fructification are intolerant to low
Temperature, even if mycelia and fructification will soften in a short time, liquefy and even rot, i.e., usually said at 4 DEG C of Conventional cryogenic
" low temperature self-dissolving ".
The uniqueness of straw mushroom " low temperature self-dissolving " proposes fresh-keeping problem.So the shelf life of straw mushroom is obviously short all the time
In other mushroom classes, so that it is cultivated and is popularized and greatly limited, also affect the performance of degrading straw economic benefit indirectly.
The fresh-keeping research of straw mushroom at present: it is confined to use for reference the postharvest technology of fruits and vegetables of broad spectrum activity physics and chemistry, such as spontaneous
The physical methods such as controlled atmosphere method, ultraviolet-sterilization, radiation sterilizing, ozone and 1- methyl cyclopropene, sodium chloride, ascorbic acid, lemon
Acid, trehalose etc..These methods have that specific aim is poor, effect is unobvious, at high cost impracticable etc..It can be said that at present
It also fails to develop the preservation by low temperature technology for being applicable in straw mushroom.The crux of the problem is not from the physiology of straw mushroom low temperature self-dissolving
Metabolic molecule mechanism is set out, and researches and develops preservation technology on this basis.
UBE2 is ubiquitin conjugation E2 enzyme, its main function is the site Lys and ubiquitin point on special selection target protein
Sub- covalent bond, and determine the specific connection type and length of ubiquitin chain, to influence the destiny of target protein.UBE2 exists
Central role is played in ubiquitin transferring path, is responsible for the diversity of ubiquitin cell signal.
Summary of the invention
Purposes the purpose of the present invention is to provide UBE2 inhibitor as food preservation by low temperature agent, this purposes
Solve in the prior art it is poor for the preservation by low temperature specific aim of straw mushroom, effect is unobvious, at high cost, unpractical technology is asked
Topic.
Purposes the present invention provides UBE2 inhibitor as food preservation by low temperature agent.
Further, the UBE2 inhibitor is NSC697923.
Further, the food is straw mushroom.
The present invention also provides a kind of low-temp antistaling methods of straw mushroom, straw mushroom are placed in the solution of UBE2 inhibitor and are soaked
Bubble, then drains away the water, is refrigerated.
Preferably, refrigerated storage temperature is 0 ~ 8 DEG C.
Preferably, soaking time 30 minutes.
Further, the UBE2 inhibitor is NSC697923.
Further, the concentration of the UBE2 inhibitor solution is 100 ~ 500 μM.
Preferably, the concentration of UBE2 inhibitor solution is 300 μM.
Present invention discover that the ubiquitination that UBEV2 is mediated leads to straw mushroom " low temperature self-dissolving ", it is a type of based on UBEV2
UBE2, the present invention inhibit ubiquitination process to weaken the principle of straw mushroom low temperature self-dissolving indirectly using the inhibitor of UBE2, in turn
Research and develop the low temperature resistant preservation technology of straw mushroom.
The present invention is matched using UBE2 inhibitor into solution, is carried out straw mushroom fructification immersion test, is found the inhibitor energy
It is enough obviously to delay low temperature to the frostbite of straw mushroom, there are the potentiality as the agent of straw mushroom preservation by low temperature.
The present invention is compared with prior art, its technical effect is that actively and apparent.It is carried out using based on UBE2 inhibitor
The low temperature resistant fresh-keeping method of straw mushroom, the invention has the following advantages that
(1) through the processed straw mushroom of the method for the present invention, it can effectively delay mushroom body to rot under Low-temperature antifreeze experiment, is flowing water, recessed
It falls into, fructification is stronger, maintains preferable exterior of commodity；
(2) easy to operate using the method for UBE2 inhibitor immersion treatment, processing cost is lower, can make the preservation by low temperature of straw mushroom
It at least extends to 2 days.
Detailed description of the invention
Fig. 1 show cold treatment induction of UBEV2 up-regulated expression, and then UBEV2 mediate ubiquitination promote straw mushroom low temperature
Self-dissolving.Wherein, (A) ubiquitination group is analysis shows that cold treatment affects protein translation；(B) Western blot analysis shows that
The UBEV2 protein expression content of straw mushroom V23 rises under cold treatment；(C) RT-PCR is analysis shows that straw mushroom VH3(VH3 bacterium under cold treatment
Strain is obtained by V23 complex mutation, and mycelium has preferable lower temperature resistance compared to V23) the mrna expression amount of UBEV2 contain
Amount decline；(D) proteomic assays show the protein expression decline of V23 under cold treatment；(E) proteomic assays show cold treatment
The protein expression of lower VH3 declines；(F) there is the interaction egg combined with UBEV2 using SDS-PAGE and the display of silver staining color method
White GST-pulldown experimental result.(G) UBEV2 interaction albumen network analysis shows that ribosomes enrichment and ubiquitin are turned into
With.(H) CO-IP experimental analysis confirms that UBEV2 is combined with the ribosomes molecular chaperone protein SSB2 of ubiquitination. (I)
UBEV2 mediates the model of ubiquitination promotion low temperature self-dissolving.The model highlights UBEV2 and acts on ribosomes molecule companion by ubiquitination
Companion's Protein S SB2 promotes its degradation, and then inhibits the effect of protein translation indirectly.
In Fig. 2, e2i represents the straw mushroom fructification photo after UBE2 inhibitor immersion treatment；H2After O represents water immersion treatment
Straw mushroom fructification photo.
Fig. 3 shows the comparison photo of the straw mushroom fructification after straw mushroom V23 and VH3 carry out cold treatment respectively.
Specific embodiment
Embodiment 1
Straw mushroom ubiquitination group analysis under cold treatment
By carrying out cold treatment 0 and 4 hour respectively to straw mushroom V23 mycelia, total protein in sample is extracted, a part is taken out and does albumen
Concentration mensuration and SDS-PAGE detection separately take part to carry out the peptide fragment richness of trypsin digestion and the ubiquitination based on antibody
Collection, then through liquid chromatograph mass spectrography identification and data analyze, obtain significant difference ubiquitination correlated expression albumen (P <
0.05 and multiple >=1.5), enrichment analysis is carried out to these ubiquitin proteins, cold treatment affects protein translation as the result is shown
(Figure 1A).
2. the Western blot of the straw mushroom UBEV2 under cold treatment is analyzed
By carrying out cold treatment 0 and 4 hour respectively to straw mushroom V23 mycelia, total protein in sample is extracted, Western blot is used
Technology carries out protein expression analysis to UBEV2, and UBEV2 protein expression content rises (Figure 1B) under cold treatment as the result is shown.
3. the RT-PCR of the straw mushroom UBEV2 under cold treatment is analyzed
By carrying out cold treatment 0 and 4 hour respectively to straw mushroom V23 mycelia, total mRNA in sample is extracted, quantitative fluorescent PCR is used
Expression technology carries out expression analysis to the UBEV2 in VH3,
The design of primers of UBEV2 is,
UBEV2-F: CTCTCCCAAGGTCGCCACTC;
UBEV2-R: GCGTGATTTCCGGGTTTGGG。
Fig. 1 C shows that RT-PCR analyzes result.RT-PCR is analysis shows that the mRNA of the UBEV2 of straw mushroom VH3 is expressed under cold treatment
Measure content decline；
Straw mushroom proteome analysis under cold treatment
By carrying out cold treatment 0 and 4 hour respectively to straw mushroom V23 and VH3 mycelia, extracts total protein and carry out iTRAQ Quantitative Western
Matter group experiment, extract sample in total protein, take out a part do determination of protein concentration and SDS-PAGE detection, separately take part into
Row trypsin digestion and label carry out chromatographic isolation after then taking each label sample of equivalent to mix, finally carry out to sample
LC-MS/MS identification and data analysis, obtain the protein expression (P<0.05 and multiple>=1.5) of significant difference.It is poor to these
M-band carry out thermal map analysis, as the result is shown under cold treatment V23 protein expression decline, and under cold treatment VH3 protein expression
Decline (Fig. 1 D and 1E).
The GST-PULLDOWN of UBEV2 is tested
UBEV2 sequence is optimized using MaxCodonTM Optimization Program (V13), is closed using full genome
GST-UBEV2 fusion is inserted into expression vector pGEX-4T-1 at and by restriction enzyme site BamHI and XhoI
In, and confirm by enzyme cutting method and sequencing the accuracy of final expression vector, finally go to respectively Top10 clone strain and
BL21 (DE3) is expressed in bacterial strain, is purified later by affinity chromatography (Glutathione resin).4 are carried out to straw mushroom V23 mycelia
It DEG C cold treatment 4 hours, extracts total protein and purified GST-UBEV2 albumen is incubated for, pass through compound with this solid phase when chromatographic column
Object can be adsorbed and separate when mixing, and separating resulting is detected (Fig. 1 F) using SDS-PAGE, silver staining.To recycling compound
Liquid chromatography mass spectrometric identification and data analysis are carried out, obtains the interaction albumen of UBEV2, and then STRING net is carried out to these interaction albumen
Network enrichment analysis (Fig. 1 G).
The co-immunoprecipitation (Co-IP) of UBEV2 is tested
Using full genome synthesis UBEV2, SSB2 (VVO_07431) and it is inserted into eukaryotic vector building expression plasmid, and is turned respectively
Change into DH5 α clone strain, transfection grade plasmid is extracted by the big pumping kit of plasmid, later turns plasmid by transfection reagent
It contaminates and is expressed in mammalian cell HEK293.The small straw mushroom V23 mycelium at present of 4 DEG C of cold treatments 4 is collected, is split using cell
Liquid lytic cell is solved, Flag monoclonal antibody is added and carries out Co-IP, experimental result is analyzed by SDS-PAGE and Western blot
(Fig. 1 H).
By above-mentioned experiment it is found that the ubiquitination that UBEV2 is mediated leads to straw mushroom " low temperature self-dissolving ".Figure I model presents cold place
The up-regulated expression of reason induction UBEV2, and then the ubiquitination of UBEV2 mediation promotes the mechanism of straw mushroom " low temperature self-dissolving ".
Embodiment 2
A method of it is low temperature resistant fresh-keeping that straw mushroom being carried out based on UBE2 inhibitor, comprising the following steps:
(1) the straw mushroom V23 for harvesting same batch selects the straw mushroom that size is similar, obviously damages without parachute-opening, nothing；
(2) NSC697923 is a kind of cell selective penetrated property UBE2 inhibitor, and the UBE2 that proportion 300ml concentration is 300 μM presses down
Preparation solution (DMSO hydrotropy)；
(3) straw mushroom is impregnated 30 minutes in UBE2 inhibitor solution, then drains away the water；
(4) by treated, straw mushroom is directly placed into 4 DEG C of progress refrigerations in 0,2,4,6,8,10,24,48 hour tests；
(5) form, color and hardness of the straw mushroom fructification of UBE2 inhibitor infiltration under cold treatment are evaluated.
As shown in Figure 2, the form of (2 ~ 48 hours) under cold treatment of the straw mushroom fructification after the infiltration of UBE2 inhibitor is full
Full, bright and hardness are strong, and everyway is significantly better than the state of control treatment.With preferable sensory evaluation effect,
Straw mushroom frost resistance significantly improves.Straw mushroom at least can be 2 days with preservation after processing.
Comparative example 1
(1) the straw mushroom V23 that will be harvested with the same batch of embodiment 1 selects the grass that size is similar, obviously damages without parachute-opening, nothing
Mushroom；
(2) using the control aqueous solution of 300ml (equivalent DMSO is added)
(3) then straw mushroom was drained away the water control aqueous solution soaking 30 minutes；
(4) by treated, straw mushroom is directly placed into 4 DEG C of progress refrigerations in 0,2,4,6,8,10,24,48 hour tests；
(5) form, color and the hardness to straw mushroom fructification under cold treatment are evaluated.
Comparative example 2
(1) the straw mushroom V23 and VH3 for harvesting same batch selects the straw mushroom that size is similar, obviously damages without parachute-opening, nothing；
(2) straw mushroom after harvesting is directly placed into 4 DEG C of progress refrigeration in 0,2,4,6,8,10,24,48 hour tests；
(3) form, color and the hardness to straw mushroom fructification under cold treatment are evaluated.
The results show that the straw mushroom fructification after the lower UBE2 inhibitor infiltration of 2 method of embodiment processing is under cold treatment
Form is fuller, color is vivider and hardness is strong, and everyway is significantly better than the state (Fig. 2) of the processing of comparative example 1.Fig. 3 knot
Fruit shows that VH3 fructification that comparative example 2 is handled under cold treatment (2 ~ 48 hours) still keeps certain vivid, vivid degree with
The state of straw mushroom fructification after the lower UBE2 inhibitor infiltration of 2 method of embodiment processing is suitable, is significantly better than at comparative example 2
The V23 fructification state of reason.There is preferable lower temperature resistance in view of VH3, the result of Fig. 3 also further confirms UBE2 inhibitor energy
Enough significantly improve straw mushroom frost resistance.
It will be apparent to those skilled in the art that can make various other according to the above description of the technical scheme and ideas
Corresponding change and deformation, and all these changes and deformation all should belong to the protection scope of the claims in the present invention
Within.
Claims (7)
- Purposes of the 1.UBE2 inhibitor as food preservation by low temperature agent.
- 2. purposes according to claim 1, it is characterised in that: the UBE2 inhibitor is NSC697923.
- 3. purposes according to claim 1, it is characterised in that: the food is straw mushroom.
- 4. a kind of low-temp antistaling method of straw mushroom, it is characterised in that: be placed in straw mushroom in the solution of UBE2 inhibitor and impregnate, so After drain away the water, refrigerated.
- 5. a kind of low-temp antistaling method of straw mushroom according to claim 4, it is characterised in that: the UBE2 inhibitor For NSC697923.
- 6. a kind of low-temp antistaling method of straw mushroom according to claim 4, it is characterised in that: the UBE2 inhibitor Solution concentration be 100 ~ 500 μM.
- 7. a kind of low-temp antistaling method of straw mushroom according to claim 4, it is characterised in that: the UBE2 inhibitor Solution concentration be 300 μM.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111423359A (en) * | 2020-03-23 | 2020-07-17 | 上海国森生物科技有限公司 | Straw mushroom UBEV2 inhibitor and screening method and application thereof |
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| CN108113983A (en) * | 2017-11-30 | 2018-06-05 | 浙江大学 | Application of the natural small molecule compounds in ubiquitin chain synthesis reaction is inhibited |
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| MING GONG， ET AL.: "A newly discovered ubiquitin-conjugating enzyme E2 correlated with the cryogenic autolysis of Volvariella volvacea", 《GENE》 * |
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| CN111423359A (en) * | 2020-03-23 | 2020-07-17 | 上海国森生物科技有限公司 | Straw mushroom UBEV2 inhibitor and screening method and application thereof |
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